title: Deciphering and engineering of the final step halogenase for improved chlortetracycline biosynthesis in industrial Streptomyces aureofaciens.
abstract: Chlortetracycline (CTC) is an important member from antibiotics tetracycline (TC) family, which inhibits protein synthesis in bacteria and is widely involved in clinical therapy, animal feeds and aquaculture. Previous works have reported intricately the biosynthesis of CTC from the intermediates in random mutants of Streptomyces aureofaciens and the crucial chlorination remained unclear. We have developed the genetic manipulation in an industrial producer, in which about 15.0g/l CTC predominated along with 1.2g/l TC, and discovered that chlorination by ctcP (an FADH2-dependent halogenase gene) is the last inefficient step during CTC biosynthesis. Firstly, the ΔctcP strain accumulated about 18.9g/l "clean" TC without KBr addition and abolished the production of CTC. Subsequently, CtcP was identified to exhibit a substrate stereo-specificity to absolute TC (4S) rather than TC (4R), with low kcat of 0.51±0.01min(-1), while it could halogenate several TC analogs. Accordingly, we devised a strategy for overexpression of ctcP in S. aureofaciens and improved CTC production to a final titer of 25.9g/l. We anticipate that our work will provide a biotechnological potential of enzymatic evolution and strain engineering towards new TC derivatives in microorganisms.
authors.affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai 20030, China.
authors.forename: Tao
authors.initials: T
authors.lastname: Zhu
authors.forename: Xueqing
authors.initials: X
authors.lastname: Cheng
authors.forename: Yuntian
authors.initials: Y
authors.lastname: Liu
authors.forename: Zixin
authors.initials: Z
authors.lastname: Deng
authors.forename: Delin
authors.initials: D
authors.lastname: You
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D002751:Chlortetracycline / Q000096:biosynthesis*; D054879:Halogenation; D060847:Metabolic Engineering; D010088:Oxidoreductases / Q000096:biosynthesis* / Q000235:genetics; D013304:Streptomyces aureofaciens / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D010088:Oxidoreductases; D002751:Chlortetracycline
keywords: Biosynthetic pathway; Halogenations; Microbial natural products; Tetracycline; Type II polyketide; Yield improvement